DHMEQ, a new NF-kappaB inhibitor, induces apoptosis and enhances fludarabine effects on chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) is a low-grade lymphoid malignancy incurable with conventional modalities of chemotherapy. Strong and constitutive nuclear factor kappa B (NF-kappaB) activation is a characteristic of CLL cells. We examined the effects of a new NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on CLL cells. Dehydroxymethylepoxyquinomicin completely abrogated constitutive NF-kappaB activity and induced apoptosis of CLL cells. Apoptosis induced by DHMEQ was accompanied by downregulation of NF-kappaB-dependent antiapoptotic genes: c-IAP, Bfl-1, Bcl-X(L) and c-FLIP. Dehydroxymethylepoxyquinomicin also inhibited NF-kappaB induced by CD40 and enhanced fludarabine-mediated apoptosis of CLL cells. Results of this study suggest that inhibition of constitutive and inducible NF-kappaB by DHMEQ in combination with fludarabine is a promising strategy for the treatment of CLL.

Protein kinase C plays a key role in the cross-talk between intracellular signalings via prostanoid receptors in a megakaryoblastic cell line, MEG-01s.

In a previous study, we characterized prostanoid and thrombin receptors expressed on a megakaryoblastic cell line, MEG-01s (Blood 78, 2328-2336, 1991). In this study, we examines the mechanism of cross-talk between intracellular Ca2+ ([Ca2+]i) and cAMP signalings through prostanoid and thrombin receptors. Addition of a thromboxane (TX)A2 mimetic (U46619 or STA2) or thrombin stimulated the formation of inositol phosphates and dose-dependently augmented a prostaglandin (PG)I2 mimetic (iloprost)- or forskolin-induced cAMP formation. 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin, to lesser extent, also augmented iloprost-induced cAMP formation. The enhancing effect of U46619 or TPA on cAMP formation was inhibited by prolonged pretreatment of the cells with TPA (2.5 microM, 24 h), but not with calmodulin-antagonists; W-7, W-5, or KN-62. The elevation of [Ca2+]i induced by thrombin, STA2 or PGE2 was significantly suppressed by pretreatment of the cells with TPA (100 nM) as well as cAMP mimetics such as dibutyryl cAMP (5 mM), forskolin (5 microM) and iloprost (1 microM). These results suggest the key role of PKC on the cross-talk between [Ca2+]i and cAMP signalings through prostanoid and thrombin receptors; PKC, which is activated with TXA2 or thrombin, concomitantly suppress further [Ca2+]i elevation and enhances the PGI2 receptor-mediated cAMP formation, which, in turn, suppress [Ca2+]i elevation.

Clinical significance of nm23-H1 proteins expressed on cell surface in non-Hodgkin's lymphoma.

The nm23 gene was isolated as a metastasis suppressor gene that exhibits low expression in high-level metastatic cancer cells. Its gene is related to the prognosis of acute myelogenous leukemia (AML) and non-Hodgkin's lymphoma (NHL). In this study, we examined the expression of nm23-H1 protein on the lymphoma cell surface of NHL. In 28 of 108 cases (25.9%), we observed > or = 20% of cell surface nm23-H1 protein expression and expression was especially high in peripheral T cell lymphomas and extranodal NK/T cell lymphomas. We also observed a significant correlation between serum nm23-H1 level and cell surface nm23-H1 expression levels. In patients with high levels of cell surface nm23-H1 expression, overall and progression-free survival rates were significantly lower than those in patients with low surface nm23-H1 expression levels. When surface nm23-H1 and serum nm23-H1 were combined, patients with high levels of both exhibited a poorer prognosis than patients with a high level of one or the other. These results indicate that in addition to serum nm23-H1, cell surface nm23-H1 may be used as a prognostic factor in planning a treatment strategy. The nm23-H1 protein appears to be intimately related to biological aggressiveness of lymphoma and, therefore, might be a molecular target of NHL treatment.

Spontaneous rupture of a left gastroepiploic artery aneurysm in a patient with autosomal-dominant polycystic kidney disease.

Autosomal-dominant polycystic kidney disease (ADPKD) has been known to be associated with a variety of vascular diseases. We present a hemodialysis patient with ADPKD who died of a massive intraperitoneal hemorrhage caused by the spontaneous rupture of a left gastroepiploic artery aneurysm. A 64-year-old male was admitted to our hospital with acute upper abdominal pain and hemorrhagic shock. An abdominal angiography showed three aneurysms and the source of hemorrhage was assumed to be the left gastroepiploic artery aneurysm. The patient died of severe metabolic acidosis and disseminated intravascular coagulation (DIC) on the second hospital day. At autopsy, there was massive bleeding into the abdominal cavity, and pathological examination of the left gastroepiploic artery aneurysm revealed a dissecting aneurysm. This is the first case describing a rupture of a gastroepiploic aneurysm in a patient with ADPKD.

Acute leukemia during pregnancy: an investigative survey of the past 11 years.

INTRODUCTION: The management of pregnant women with acute leukemia is usually challenging. We collected data concerning pregnant women with acute leukemia in the Kanagawa area in Japan. METHODS: A questionnaire was sent to 24 institutions in the Kanagawa area. RESULTS: Data were obtained for 11 patients, median age of 31 years (range, 20-36). Eight patients had acute myeloid leukemia and three had acute lymphoblastic leukemia. Six patients were diagnosed in the first trimester of pregnancy, one in the second trimester, and four in the third trimester. Five of six patients diagnosed in the first trimester had abortions before chemotherapy, and one had an elective abortion after receiving chemotherapy. All patients diagnosed in the second or third trimester delivered live infants. Of the six patients diagnosed in the first trimester, two died of recurrent leukemia, and four remained in remission. Of the five patients diagnosed in the second or third trimester, four achieved complete remission and remained in remission. One patient died of sepsis 4 days after cesarean section. CONCLUSIONS: Careful surveillance and monitoring of the fetus and close co-operation among hematologists, gynecologists, and pediatricians are essential to successfully treat pregnant women with acute leukemia.

Inhibition of 20-kDa myosin light chain exchange by monoclonal antibodies against 17-kDa myosin light chain.

Two anti-17,000 Da myosin light chain (LC17) monoclonal antibodies (MM2 and MM10), which increase the actin-activated Mg(2+)-ATPase activity of dephosphorylated smooth muscle myosin, inhibited the exchange of the 20,000 Da regulatory light chain of myosin (LC20). MM2, which shows higher potency of activation of ATPase activity, inhibited the exchange more extensively than MM10, suggesting that there is a correlation between the activation of ATPase activity and the inhibition of the LC20 exchange. The inhibition of the exchange was observed for intact myosin and heavy meromyosin but not subfragment 1, suggesting that the heavy chain at the head-rod junction is involved in the inhibition of LC20 exchange by anti-LC17 antibodies. Alternatively, the interaction between the two heads of the myosin molecule may influence the inhibition of LC20 exchange. These results suggest that LC20 interacts with both LC17 and the heavy chain, and the interaction between LC20 and LC17 is involved in the activation of actin-activated ATPase activity of smooth muscle myosin.

Alteration of the enzymatic properties of smooth muscle myosin by a monoclonal antibody against subfragment 2.

A monoclonal antibody against subfragment 2 (S-2) of smooth muscle myosin, designated MM-9, was generated and characterized. MM-9 potently inhibited subfragment 1 (S-1) release by papain proteolysis of myosin, suggesting that the epitope of MM-9 is at or very close to the S-1/S-2 junction. The depression of Ca2(+)- and Mg2(+)-ATPase activities of myosin at low ionic strength was significantly reduced by MM-9. MM-9 increased the acto dephosphorylated HMM ATPase activity about 3-fold. On the other hand, the antibody had no effect on the KCl-dependence of viscosity of monomeric myosin. These results suggest that the folding of the myosin rod is not the direct determinant of enzymatic activity, and that the subtle conformational change at the S-1/S-2 junction (head-neck region) plays a critical role in determining enzymatic activities.

The inhibitory effects of okadaic acid on platelet function.

Okadaic acid (OA), a potent inhibitor of protein phosphatases type 1 and type 2A, inhibited thrombin-induced platelet aggregation (IC50 = 0.8 microM), [14C]serotonin release and increase in intracellular Ca2+ ([Ca2+]i) in the same dose dependence. In the absence of thrombin OA increased the phosphorylation of 50-kDa protein and 20-kDa myosin light chain (MLC20). The 50-kDa protein phosphorylation was accomplished within a shorter time period and at a lower concentration than was the MLC20. OA decreased the thrombin-induced phosphorylation of 47-kDa protein and MLC20, although phosphorylation of MLC20 reincreased at higher concentrations of OA (5-10 microM). Since type 2A phosphatase is more sensitive to OA than type 1, these results suggest that type 2A phosphatases are involved in the regulation of Ca2+ signaling in thrombin-induced platelet activation.

The role of apoE in inhibitory effects of apoE-rich HDL on platelet function.

Apolipoprotein E- (ApoE-) rich high-density lipoprotein (HDL), which was prepared from the bound fraction of normolipemic volunteers on heparin-Sepharose and from a hyperalphalipoproteinemic patient, potently inhibited aggregation of human platelets in a dose-dependent fashion. Dimyristoyl phosphatidylcholine liposome with apoE (apoE.DMPC) also inhibited platelet aggregation, and incubation of washed platelets with apoE.DMPC resulted in the release of cholesterol into the supernatant in a time- and dose-dependent manner. These results suggest that apoE plays a major role in the inhibitory effect of apoE-rich HDL in platelet function, presumably due to the release of cholesterol from the plasma membrane.

Purification and partial characterization of CD9 antigen of human platelets.

CD9 antigen (p24) was purified from human platelets and partially characterized. The yield was 75 micrograms from 10 units of platelet concentrates. p24 (38,000 copies/platelet) has intramolecular disulfide bond(s) and, in SDS-PAGE, consists of major 24-kDa molecule and minor 26- to 31-kDa molecules. The N-terminal sequence of p24, PVKGGTKXIKYLLFGFNFIF, indicates that the protein has not previously been characterized and amino terminus (position 12-20) is hydrophobic.

The platelet activation induced by wheat germ agglutinin.

In human platelets, wheat germ agglutinin (WGA) induced serotonin release without cell agglutination. WGA induced the phosphorylation of both 40-kDa and 20-kDa proteins in a parallel manner, and at least, the phosphorylation of 40-kDa protein was preceded by transient formation of endogenous diacylglycerol (DG) accompanied by a decrease in phosphatidylinositol (PI). Both phosphorylation of these two proteins and serotonin release were inhibited by prior treatment of platelets with dibutyryl cyclic AMP, W-7, or TMB-8. These results suggest that both phosphatidylinositol turnover and Ca2+ mobilization play an essential role in WGA-induced platelet activation.

Positive correlation between prolonged potentiation of binding of double-stranded oligonucleotide probe for the transcription factor AP1 and resistance to transient forebrain ischemia in gerbil hippocampus.

Gel retardation electrophoresis revealed that binding of a radiolabelled double-stranded oligonucleotide probe for the nuclear transcription factor activator protein-1 was markedly potentiated in the CA1 and CA3 subfields and the dentate gyrus of the hippocampus of the gerbils with transient forebrain ischemia for 5 min, which is known to induce delayed death of pyramidal neurons exclusively in the CA1 subfield. The potentiation was transient in the vulnerable CA1 subfield, but persistent up to 18 h in the resistant CA3 subfield and dentate gyrus. However, no significant alteration was detected in endogenous levels of cyclic AMP response element binding protein phosphorylated at serine133 in these three different hippocampal structures 3 h after the reperfusion. On the other hand, hypothermia during ischemia which is known to protect the CA1 subfield against ischemic damages, led to a prolonged elevation of the activator protein-1 binding up to 9 h after the reperfusion in this vulnerable subfield at least in part through expression of c-Fos protein. Moreover, activator protein-1 binding was significantly elevated in the CA1 subfield up to 12 h after forebrain ischemia for 2 min which is shown not to induce marked damages to the vulnerable subfield. These results suggest that prolonged elevation of DNA binding activity of activator protein-1 may be responsible for molecular mechanisms underlying the unique vulnerability and/or resistance of particular subfields to a transient ischemic insult in the gerbil hippocampus.

Modification of Na+/H+ exchanger in human platelets by 1,2-dioctanoylglycerol, a protein kinase C activator.

Protein kinase C activation in human platelets has a modulatory role in maintaining intracellular pH (pHi), by adjusting pHi at a particular value (7.22). Changes in pHi induced by protein kinase C appeared to be dependent upon the difference between H+ efflux catalyzed by the Na+/H+ exchanger and H+ production. The pHi recovery after acid loading was significantly facilitated by protein kinase C activation. Analysis of the rate constant for pHi recovery suggested that the turnover rate or the apparent affinity of the Na+/H+ exchanger for H+ was increased. Protein kinase C also decreased the Km value of the Na+/H+ exchanger for extracellular Na+. Thus, it is suggested that the role of protein kinase C in platelet pHi regulation is dual, adjusting the pHi value at a certain setpoint on the one hand, and increasing the rate constant of the Na+/H+ exchanger on the other.

Evaluation of thrombopoiesis in thrombocytopenic disorders by simultaneous measurement of reticulated platelets of whole blood and serum thrombopoietin concentrations.

To evaluate thrombopoiesis in thrombocytopenic disorders, we simultaneously determined reticulated platelet counts in whole blood by FACScan flow cytometry and serum thrombopoietin (TPO) concentrations by a sensitive sandwich ELISA. The subjects were 40 healthy volunteers and 45 thrombocytopenic patients. In idiopathic thrombocytopenic purpura (ITP), the percentage of reticulated platelets was significantly elevated (5.61 +/- 2.02%: mean +/- SD) relative to normal controls (2.17 +/- 0.90%), but serum TPO concentrations (1.91 +/- 1.27 fmol/l) did not differ significantly from the normal range (1.43 +/- 0.62 fmol/l). The patients with aplastic anemia (AA) had decreased reticulated platelet counts and markedly increased serum TPO concentrations (13.65 +/- 10.64 fmol/l). In thrombocytopenic patients with liver cirrhosis (LC), the absolute number of reticulated platelets (1.65 +/- 1.11 x 10(9)/l) decreased similarly that in AA. However, serum TPO concentrations (1.38 +/- 0.50 fmol/l) did not increase in contrast to AA. Our findings suggested a possible dual mechanism of thrombocytopenia in LC; that is, thrombocytopenia in LC results from the decreased TPO production primarily in the liver adding to an increase in platelet sequestration in the spleen.

The novel variants of mb-1 and B29 transcripts generated by alternative mRNA splicing.

The Ig-alpha/Ig-beta heterodimers encoded by mb-1 and B29 genes, respectively, are crucial for the constitution of the B-cell receptor (BCR). We report here novel variants of mb-1 and B29 transcripts produced by alternative mRNA splicing. The proteins encoded by these variants are predicted to conserve transmembrane and cytoplasmic portions of Ig-alpha and Ig-beta but lack a part of the extracellular portions containing cysteine residues which are required for intramolecular and intermolecular S-S bonds. Transfection studies revealed that the variant mb-1 and B29 did not contribute to the BCR expression on cell surfaces. Although peripheral B cells contain small amounts of the variant mb-1 and B29 transcripts, treatment with an anti-IgM antibody, LPS or IL-4 induces a significant increase in amounts of the variant transcripts. These observations suggest that B-cell activation induces alternative splicing of mb-1 and B29 transcripts which encode proteins unable to constitute the BCR.

Effects of various inhibitors on platelet activation induced by TP 82, a CD 9 monoclonal antibody.

TP 82, a monoclonal antibody against CD 9 antigen, induced human platelet activation at concentrations higher than 0.4 microgram/mL in terms of aggregation, release of intracellular granule contents, production of arachidonic acid metabolites, and elevation of the intracellular Ca2+ concentration. The effects of a competitive inhibitor of ADP, acetylsalicylic acid, EGTA, and GRGDSP which blocks fibrinogen binding to IIb/IIIa complex suggested that each of released ADP, thromboxane A2, extracellular Ca2+, and close cell contact acts together to potentiate platelet activation induced by TP 82. While each of these inhibitors severely suppressed platelet activation induced by lower concentrations of the antibody (less than or equal to 0.8 microgram/mL), that induced by higher concentrations (greater than or equal to 3.2 micrograms/mL) was only partially blocked. Intracellular Ca2+ elevation was totally dependent upon the production of thromboxane A2, regardless of the antibody concentrations.

Effects of five anion channel blockers on thrombin- and ionomycin-activated platelet functions.

The inhibitory effects of anion channel blockers were evaluated on aggregation, intracellular Ca2+ rises, and the production of arachidonic acid metabolites in human platelets. Inhibitors included five anion channel blockers: phloretin, probenecid, pyridoxal phosphate, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). The degree of inhibition by each of these agents was dose-dependent on thrombin-activated platelet function. These agents generally had no significant inhibitory effects on ionomycin-activated platelet functions. It is suggested that anion mobilization plays a major role in the receptor-mediated activation of platelet functions, but only a minor role in Ca2+ ionophore-induced platelet activation. It is also suggested that several agents may have properties unrelated to anion channel blockers. Phloretin may be a selective cyclooxygenase inhibitor, and probenecid may inhibit phospholipase A2. DIDS and SITS may interfere with certain aggregation-inducing mechanisms.

Effects of the prior activation of protein kinase C on human platelet activation induced by thrombin.

Effects of the prior activation of protein kinase C (PKC) on the responses induced by thrombin were studied using human blood platelets, in which PKC is abundantly expressed. At a concentration of 25 nM, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) induced little aggregation or release by itself but selectively elicited PKC activation in a time-dependent manner, as monitored by 47-kDa protein phosphorylation. Increases in the intracellular Ca2+ concentration of human platelets caused by thrombin, at any concentration, were markedly inhibited by the prior addition of 25 nM TPA in a time-dependent manner. However, the effects of TPA on platelet aggregation and secretion induced by thrombin varied, depending upon the agonist concentration; the PKC activator markedly enhanced the aggregation and secretion induced by lower concentrations of thrombin but had a tendency to weakly inhibit those induced by higher concentrations of thrombin. TPA enhanced the ionomycin effect upon aggregation and release without obvious effects on the ionophore-induced Ca2+ mobilization, suggesting that PKC potentiates platelet function by increasing intracellular sensitivity to Ca2+. There was a good time-dependent correlation of the TPA effects among the three parameters, namely, the phosphorylation of the 47-kDa protein, inhibition of the [Ca2+]i increase induced by thrombin, and enhanced ionomycin effects in aggregation and release. It is most likely that the final responses of agonist-activated platelets depend upon the balance of two factors induced by PKC; inhibition of Ca2+ signals and enhancement of intracellular sensitivity to Ca2+.

Suppression of humoral immunity by monoclonal antibody to CD79b, an invariant component of antigen receptors on B lymphocytes.

CD79b is an invariant component of antigen receptors on B lymphocytes. Previous data have suggested that monoclonal antibody (mAb) to CD79b would introduce negative signals into B lymphocytes and suppress humoral immunity. We tested this hypothesis in this study using in vitro assay systems, and revealed that anti-CD79b mAb effectively suppressed the antibody response to a T-cell dependent antigen. The speculated mechanisms for this immunosuppression were: (i) down-modulation of antigen receptors, (ii) inhibition of B lymphocyte differentiation, and (iii) induction of B lymphocyte unresponsiveness. Of these three, we confirmed that the first two were actually induced by anti-CD79b mAb treatment, whereas the in vitro system could not induce the unresponsiveness of B lymphocytes.

Tyrosine phosphorylation of proteins in primary human myeloid leukemic cells stimulated by macrophage colony-stimulating factor: analysis by disease type and comparison with normal human hematopoietic cells.

We investigated tyrosine phosphorylation of proteins in primary human leukemic cells stimulated by macrophage colony-stimulating factor (M-CSF) in 60 patients with acute myeloid leukemia (AML) and 5 patients with chronic myelomonocytic leukemia and compared the findings for leukemic cells with those of normal human monocytes and bone marrow immature hematopoietic cells. M-CSF induced tyrosine phosphorylation of p140-200, p110, p60, p44, and p42 frequently, and that of p95 and p55 less frequently, in primary myeloid leukemic cells, whereas M-CSF-induced phosphorylation of proteins was not detected in the normal human hematopoietic cells tested. Of these phosphoproteins, p140-200 was phosphorylated in all patients who responded to M-CSF and was considered to be almost identical to Fms, a product of the c-fms proto-oncogene. M-CSF-induced tyrosine phosphorylation was observed frequently (89%) in AML of French-American-British class M4 and infrequently in all other subtypes of AML, including M5. In chronic myelomonocytic leukemia, M-CSF-induced protein phosphorylation was prominent in blast crisis but was not detected in the chronic phase. Both bone marrow immature cells and mature monocytes showed low responsiveness to M-CSF. These findings for responsiveness to M-CSF were correlated with the amount of Fms in each type of cell. We also identified tyrosine phosphorylation of Vav, Shc, and extracellular signal-regulated kinase by M-CSF in some cases. These findings clarified an M-CSF-specific pattern of protein tyrosine phosphorylation and the responsiveness to M-CSF of primary human myeloid cells. Particularly, enhanced phosphorylation responses to M-CSF and increased amounts of Fms protein were observed in restricted human hematopoietic cells with a premature myelomonocytic character.